Conserved cellular patterning in the mesophyll of rice leaves

Abstract The mesophyll cells of grass leaves, such as rice, are traditionally viewed as displaying a relatively uniform pattern, in contrast to the clear distinctions of palisade and spongy layers in typical eudicot leaves. This quantitative analysis of mesophyll cell size and shape in rice leaves reveals that there is an inherent pattern in which cells in the middle layer of the mesophyll are larger and less circular and have a distinct orientation of their long axis compared to mesophyll cells in other layers. Moreover, this pattern was observed in a range of rice cultivars and species. The significance of this pattern with relation to potential photosynthetic function and the implication of the widespread use of middle layer mesophyll cells as typical of the rice leaf have been investigated and discussed.

mesophyll of rice leaves.The manuscript technically sounds well and shows high novelty.It is well written and shows the need for minor linguistic adjustments.In this regard, the needed adjustments are highlighted in "Minor comments" section.The use of past verbal tense is needed when reporting the findings of the study.The first voice form of the sentence shall be avoided and the impersonal form shall be adopted instead.The references used in both Introduction and Discussion sections shall be up-to-date (2010 and onwards).The Abstract section outlines clearly the problematic, aims, methodology and findings of the current study while reporting the main conclusions aroused.The Introduction section is well structured and aiming and underlines appropriately the whole subject under study.The aims of the study are also clear and understood.The Materials and methods section is clear, well written, and encloses all the information related to the adopted methodology, and statistical analysis.The Results section shows a correct statistical representation and an adequate scientific analysis of the findings.Similarly, the Discussion section compared the current study's findings with those of previously published studies in literature.Authors highlighted the main strength point of their study and its limitations; they further suggested related research being based on the raised assumptions and simulations.My comments and queries for authors are detailed below in "Minor comments" section.

Reviewer #2:
In this study, Jen et al. revealed that the size and shape of mesophyll cells are different depending on the layers in rice leaves, which are traditionally viewed as displaying a relatively uniform pattern.This pattern is confirmed by conducting a quantitative analysis based on microscopy images and computational modeling in the leaves of different rice cultivars.Notably, the author suggested that the significance of this pattern could be related to photosynthetic activities.I believe this study has a novelty and might help determine photosynthetic efficiency in grass plants.Overall, the article was well-written, and the experiments were well-designed.I could not find a significant mistake, and this manuscript could be accepted as it is.

DECISION LETTER-ROUND 2
November 1, 2023 Dr. Jen Sloan University of Sheffield Sheffield, N/A United Kingdom MSID: 2023-01290R1 MS TITLE: Conserved cellular patterning in the mesophyll of rice leaves Dear Dr. Jen Sloan: I am pleased to inform you that your manuscript "Conserved cellular patterning in the mesophyll of rice leaves" has been accepted for publication in Plant Direct.
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We thank Reviewer #1 for their kind comments, and for the care they have taken with their extremely helpful recommendations throughout the text.We have taken all of this on board, as detailed below.

Minor comments:
1-The use of past verbal tense is needed when reporting the findings of the study.2-The first voice form of the sentence shall be avoided and the impersonal form shall be adopted instead.

3-The references used in both Introduction and Discussion sections shall be up-to-date (2010 and onwards).
Thank you for this suggestion.We have taken on this advice and added more up to date references in a number of places in the text.In some places, we have not removed the older references, as we felt that including them helped to show that the conclusions mentioned are long-held.However, if the editor would prefer to remove the older references, then this is not a problem.In addition to the suggested additions, we have included (Terashima et al., 2016;Gotoh et al., 2018;Holloway-Phillips, 2019) in line 52.

Reworded as follows:
"For example, in recent work from the Fleming group, a computational model of rice leaf photosynthesis, eLeaf was developed, which incorporated a 3D structure of IR64 rice leaves abstracted from image data of rice leaves at various resolutions using a range of approaches (Xiao et al., 2023)."10-1.Introduction: Pages 3-4, lines 75-80: "To start... leaf": Same recommendation as in the previous comment.

Reworded as follows:
"To start to address this issue, a quantitative analysis of the mesophyll was performed in both IR64 and a range of other rice cultivars and species.The results, reported here, indicate that the rice leaf mesophyll had a more complex and ordered cellular architecture than previously acknowledged.A 2D modelling approach was then utilised, and the significance of this particular mesophyll structure to the photosynthetic performance of the rice leaf was explored."

Reworded as follows:
"To investigate whether there was a pattern of mesophyll size and shape, mesophyll cells were assigned to different layers (1 to 5) within the leaf (Fig. 1A)." 22-3.Results: Page 7, lines 173-175: "We assigned... and shape": Same recommendation as in the previous comment.

Reworded as follows:
"Mesophyll cells were assigned to the different cell layers, then the cells were analysed for a range of quantitative parameters linked to size and shape (Fig. 1B-F)."23-3.Results: Page 8, lines 183-186: "Rice... parameter": Same recommendation as in the previous two comments.

Reworded as follows:
"Rice mesophyll cells are characterised by being highly lobed (Sage and Sage 2009), therefore this trait was also quantified, using a calculation of the ratio of actual cell perimeter to the minimal energy circumference of each cell as a measure of this parameter.": "To investigate... mesophyll": Same recommendation as in the previous comments.

Reworded as follows:
"To investigate this, measurements were made of the angle of the long axis of individual mesophyll cells in each layer of the mesophyll (Fig. 1F)." : "These data... 20-50{degree sign}": Kindly adopt the past verbal tense for these sentences.

Reworded as follows:
"These data generally substantiated the impression obtained from Fig 1E, i.e., the Layer 3 cells had a long axis which was predominantly aligned with the horizontal axis, which was also true for Layer 2 and Layer 4 cells.This equates to the mediolateral plane of the rice leaf, at right angles to the long axis of the leaf.The sub-epidermal mesophyll cells in Layer 1 displayed a greater variation in cell axis orientation, and Layer 5 cells were mostly orientated between 20-50°."26-3.Results: Page 8, lines 202-206: "Having... O. meridionalis": Kindly avoid the first voice form of these sentences and adopt the impersonal form instead.

Reworded as follows:
"In order to establish whether this pattern of mesophyll cell size and shape in IR64 leaves was reflected in the wider rice family, the mesophyll was studied in a range of rice varieties, including three cultivated Oryza sativa Indica variants (MRQ76, MR220 and Malinja), and three wild species (O. latifolia, O. punctata and O. meridionalis)."Also changed 'show' to 'showed' in line 201.

Reworded as follows:
"To investigate how the pattern of mesophyll cell size revealed by this analysis might influence leaf photosynthetic performance in terms of basic light absorption and carbon assimilation rate, an initial 2D modelling approach was utilised."Kindly adopt the past verbal tense for all these sentences.

Reworded as follows:
"Model 1 was most representative of mesophyll described in this study, with five layers, in which the middle layer (Layer 3) was characterised by having relatively large cells (Fig. 7A).Model 2 had the same number of layers as Model 1, but the cells were all uniform and relatively small (Fig. 7B).
Consequently, the Model 2 leaf was slightly thinner than Model 1. Models 3 and 4 were made up entirely of relatively large cells with four and five layers, respectively (Fig. 7C,D).Model 4 was therefore slightly thicker than Models 1, 2 and 3.Each cell was modelled to have a proportion of plastid inversely proportional to cell size and a proportion of cytosol proportional to cell size, reflecting the findings of Sage and Sage (2009).Cell wall thickness and mitochondrial size were the same in all models.As a consequence of these parameters and packing, Models 1, 2 and 4 had the same plastid and cytosol volumes (Fig. 7E), but differed in their leaf thickness.Model 3 had the lowest plastid and cytosol volume.The amount of cell wall in contact with the air (Smes) was very similar in Models 1 and 2, lowest in Model 3 and intermediate in Model 4 (Fig. 7E)."Reworded as follows: "The analysis reported here indicated that rice leaves from a range of genotypes and variants display a conserved pattern of cellular architecture.In particular, mesophyll cells in the middle (Layer 3) of the leaves were larger than mesophyll cells in other layers (Fig. 1B, Fig. 3, Fig. 4A), had the lowest degree of circularity (Fig. 1C, Fig. 4B, Fig. 5) and tended to have a cell long axis oriented orthogonal to the longitudinal axis of the leaf, thus aligning with the medio-lateral plane of the leaf (Fig 1E,Fig. 6).A distinctive pattern of cell axiality was also observed in the most adaxial mesophyll layer (Layer 1) where cells displayed a much wider range of long axis orientation than cells in the other layers of the mesophyll.These cells were also most likely to be ranked as the least lobed (Fig. 1D, Fig. 4C)."This sentence is specifically referring to the long-established belief that monocot mesophyll is uniform, hence we have used older references as well as Beck, 2010 andPyke, 2012.Have also updated the reference in line 311 to Terashima et al., 2016;Holloway-Phillips, 2019. 37-4.Discussion, Rice mesophyll displays a conserved pattern of size and shape: Page 13, line 325: "To make... approach": Kindly avoid the first voice form of the sentence and adopt the impersonal form instead.

Reworded as follows:
"To make an initial analysis of this problem a modelling approach was taken."38-4.Discussion, Rice mesophyll displays a conserved pattern of size and shape: Page 13, line 327: Kindly adjust as follow: "led".Done 39-4.Discussion, Rice mesophyll displays a conserved pattern of size and shape: Page 13, lines 334-335: "At present... speculate": Kindly remove this sentence.

Done
Reviewer #2: In this study, Jen et al. revealed that the size and shape of mesophyll cells are different depending on the layers in rice leaves, which are traditionally viewed as displaying a relatively uniform pattern.This pattern is confirmed by conducting a quantitative analysis based on microscopy images and computational modeling in the leaves of different rice cultivars.Notably, the author suggested that the significance of this pattern could be related to photosynthetic activities.I believe this study has a novelty and might help determine photosynthetic efficiency in grass plants.Overall, the article was well-written, and the experiments were well-designed.I could not find a significant mistake, and this manuscript could be accepted as it is.
We thank Reviewer #2 for their kind comments.
-Reviewer comments:AUTHORS' RESPONSEReggie Millwood and Gustavo MacIntosh, Thank you for your comments, and for those of the reviewers.I have detailed below the changes we have made in response to them.Jen Sloan - -Editor comments: Please add the figure number for each figure in your figure file.Done Please change the reference to Supp.Fig. 3 to Supp.Fig. S3 (see line 207).Done Please change the reference to Supp.Fig. 4 to Supp.Fig. S4 (see line 233).Done - -Reviewer comments: Reviewer #1: Comments to the Author: Title: Conserved cellular patterning in the mesophyll of rice leaves Overview and general recommendation: 4-Abstract: Page 2, lines 27-30: "In this paper... layers": Kindly write the sentence in the past verbal tense form.Moreover, kindly avoid the first voice form of the sentence and adopt the impersonal form instead. Done 5-Abstract: Page 2, line 30: Kindly replace "is" by "was".Done 6-Abstract: Page 2, line 33: Kindly replace "are" by "were".Done 7-1.Introduction: Page 2, lines 44-47: "These parameters... mesophyll": These statements lack reliable sources (references); accordingly, kindly provide them.Done -added (Baillie and Fleming, 2020; Theroux-Rancourt et al. 2021) in line 45-46 and (Baillie and Fleming, 2020) in line 48.8-1.Introduction: Page 3, lines 52-56: "In addition... mechanics": Both sentences repeat the same information; accordingly, kindly reformulate.Deleted first sentence.9-1.Introduction: Page 3, lines 68-71: "For example... approaches": Kindly avoid the first voice form of the sentence and adopt the impersonal form instead.
35-4.Discussion, Rice mesophyll displays a conserved pattern of size and shape: Page 12, lines 288-290: "These observations... the leaf": The references used for this statement are very old; accordingly, kindly replace them by more recent ones (2010 and onwards).Replaced with Beck, 2010, Pyke, 2012 Also replaced 'Our data' with 'The data presented in this article' in line 298 to avoid the first person voice.36-4.Discussion, Rice mesophyll displays a conserved pattern of size and shape: Page 12, line 302: Same recommendation as in the previous comment regarding the first reference.
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